Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
J Am Chem Soc. 2011 Apr 20;133(15):5676-9. doi: 10.1021/ja1117396. Epub 2011 Mar 25.
Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a fluorescence resonance energy transfer (FRET)-based readout, has often been a limiting factor. Here we show that a kinase-inducible bimolecular switch consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility. Specifically, we demonstrate that bimolecular switches designed to sense protein kinase A (PKA) or protein kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges in comparison with their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.
实时跟踪活细胞中的激酶活性揭示了将信号信息编码为时空活性模式的新方式，并为筛选激酶调节剂开辟了新途径。然而，通常与基于荧光共振能量转移 (FRET) 的读出相关联的激酶活性检测灵敏度常常是一个限制因素。在这里，我们展示了一种由感兴趣的激酶的底物和磷酸氨基酸结合域组成的激酶诱导双分子开关可以设计用来感知不同的激酶活性，并与各种读出方式耦合，从而可以提高灵敏度和多功能性来检查动态激酶活性。具体来说，我们证明了设计用于感应蛋白激酶 A (PKA) 或蛋白激酶 C (PKC) 活性的双分子开关可以打开 FRET 以及生物发光信号。值得注意的是，与单分子对应物相比，基于 FRET 的传感器具有更大的动态范围；用于 PKA 和 PKC 的新型生物发光报告器具有高灵敏度和独特的检测基础激酶活性的能力，应该能够在激酶活性的体内成像和高通量化合物筛选中实现新的应用。因此，这种可推广的设计推进了激酶活性检测的分子工具包，并为在各种生物系统中进行多功能和灵敏的激酶活性检测提供了一种手段。
